Aqueous dispersions of nanometric or micrometric particles for encapsulating chemical compounds

ABSTRACT

The present invention relates to a composition comprising an aqueous dispersion of particles (p) of mean hydrodynamic diameter between 50 and 5000 nm, the said particles containing in association: (A) polymers based on cyclodextrin units, comprising on average at least 4 cyclodextrin units within their structure; and (B) macromolecules of polysaccharides comprising groups G capable of forming inclusion complexes with the cyclodextrins present in the structure of the said polymers (A), with an average number of groups G per polysaccharide macromolecule at least equal to 3, the said compounds (A) and (B) being water-soluble in the isolated state. The invention also relates to the method of preparation of these compositions, as well as their use in order to achieve the encapsulation of compounds such as active substances.

The present invention relates to aqueous dispersions of nanometre ormicrometre dimensions, suitable for the encapsulation of chemicalcompounds and especially useful for carrying out the vectorisation ofactive substances at the level of cells or of target tissues, especiallyin pharmaceutical or cosmetic applications.

In the course of the last twenty years, numerous systems forvectorisation of active substances have been developed. In a generalmanner, the principle of vectorisation consists of trapping an activesubstance within a particle of micrometric or submicrometric size whichcan be administered in a subject to be treated (particularly byinjection or ingestion) and by choosing the said particle in such amanner that it releases the active substance within a cell or a targetedtissue.

Within this context, development has been carried out in particular onthe encapsulation of active substances by liposomes (lipid bilayerssurrounding an aqueous core) or even by nanocapsules (polymer shells ofsub-micrometer size containing a phase, generally oily, including theactive substance).

More specifically, systems such as nanospheres or microspheres have alsobeen developed, namely particles based on biocompatible andbiodegradable polymers (for example polylactic acid or polycaprolactone)playing the role of a matrix capable of immobilising active substancesand releasing them progressively within an organism where this system ofencapsulation is introduced.

These systems of the nanosphere or microsphere type generally have aquite good capacity for vectorisation of active substances. However,these vectors of active substances currently have a major drawback. As amatter of fact, the processes of preparation of systems of this typewhich are currently known implement intermediate steps which generallyimply the use of organic solvents or non-biocompatible surfactants whichhave to be eliminated. The elimination of these solvents and/orsurfactants is generally long and expensive (carrying out dialysis,ultracentrifugation, . . . ), and it is rarely complete. Therefore, atthe end of such purification steps trace amounts of solvents and/orsurfactants generally remain, which is likely to create problems interms of toxicity (for example if the solvent remaining in the state oftraces is a solvent such as dichloromethane) and/or to degrade theactive substances immobilised in the particles (traces of organicsolvents can be for example denature fragile molecules of the peptide orprotein type).

Now, the inventors have unexpectedly discovered that it is possible toproduce aqueous dispersions of particles of nanosphere or microspheretype, suitable for vectorisation of active substances, without having touse organic solvents or surfactants in this connection.

More precisely, the inventors have discovered that, if one makes inaqueous medium a mixture of specific water-soluble compounds, namely (1)polymers comprising cross-linked cyclodextrin units and (2)macromolecules carrying groups able to form inclusion complexes with thecyclodextrins (such as, for example, polysaccharides substituted byaliphatic chains), one may observe under specific conditions, thespontaneous formation of a dispersion of particles of mean hydrodynamicdiameter between 50 and 5000 nm, if subject to suitable choice of theused compounds (1) and (2) used.

Associations of polymers comprising cyclodextrin units andpolysaccharides substituted by aliphatic chains in an aqueous mediumhave already been described. In this connection, it is especially knownthat, when polymers comprising cyclodextrin units and polysaccharidessubstituted by aliphatic chains are present in an aqueous medium, theformation of inclusion complexes between the aliphatic chains of thesubstituted polysaccharides and the cyclodextrin units of the polymersoccurs, which leads to an association of the polymers and thepolysaccharides.

However, until now, this association in an aqueous medium has alwaysbeen specifically described as being capable of leading only to theformation of two types of associative systems, namely:

-   -   (I) a homogeneous single-phase comprising associated polymers        and polysaccharides dissolved in the aqueous medium; or    -   (II) a non-emulsifiable two-phase system comprising a first        phase which is rich in polymers, dense and viscous (a so-called        “gel”) and a second homogeneous phase which is less dense and        viscous (a so-called “supernatant”) which is obtained by a        mechanism known as “associative phase separation” which has in        particular been described in a publication by the ACS, edited        by C. McCormick (Symposium Series, No. 780, chapter 4,        “Stimuli-Responsive Water-Soluble and Amphiphatic Polymers”, C.        Amiel et al, 2000).

In this connection, it is also known that certain associations (polymerscomprising cyclodextrin units/polysaccharides substituted by aliphaticchains) lead, in an aqueous medium, exclusively to the formation of asystem of type (I) regardless of the respective concentrations ofpolymers and polysaccharides (associations known here as type a_(I)).The type (II) system can only be obtained for other particularassociations, known here as type a_(II). In the following description,these specific associations of a,, type will be designated by thegeneric term of auto-associative system “of a type which can lead, undercertain concentration conditions, to an associative phase separation”.

As regards the associations of type all defined above, it should beemphasised that the associative phase separation can only be observedunder specific concentration conditions, corresponding to a relativelylimited range on a ternary water/polymer/polysaccharide diagram. It hashitherto been generally acknowledged that, in this range of specificconcentrations, it was only possible to observe phenomena of theassociative phase separation type leading to the formation of a systemof type (II) and that, outside this range, it was only possible toobserve the formation of a system of type (I).

The present invention is based on the entirely unexpected discovery ofthe possibility of forming a third type of system when starting fromassociations of type a_(II). As regards these specific associations, thework of the inventors has actually allowed to evidence that a specificrange of concentrations exists wherein a direct mixture of water,polymers based on cyclodextrin units, and polysaccharide grafted withaliphatic chains, leads to the formation of a dispersion of particlesbased on associated polymers and polysaccharides, having a meanhydrodynamic diameter at least equal to 50 nm, and most often havingvery good stability. More generally, the inventors have discovered thatsuch dispersions of particles can be obtained by the direct mixture ofwater, polymers based on cyclodextrin units and macromolecules carryinggroups likely to form inclusion complexes with the cyclodextrins.

In this connection, the inventors have established that the dispersionthus produced constitutes a metastable system, and that only the systemsof type (I) and (II) are thermodynamically stable systems. Thus, if adispersion as discovered by the inventors is subjected to sufficientlysubstantial shear conditions, a development towards a system of type(II) is generally observed. In this context, the inventors have howeversurprisingly discovered that the stability of the metastable dispersionproduced is unexpectedly high, which especially makes it possible tocontemplate its storage, dilution, or even its integration in complexcompositions, for example in pharmaceutical or cosmetic compositions.

On the basis of the inventors' work, it has also been evidenced that theparticles formed within the obtained metastable dispersions areespecially suitable for the encapsulation of chemical compounds, andparticularly for the vectorisation of active substances, with aneffective and quantitative integration of the chemical compounds. Inthis scope, the inventors have especially evidenced that these particlesallow to achieve a release of the active substances in a much morecontrolled manner than when using most of particles of nanosphere ormicrosphere type of the prior art, for which an immediate release of asignificant fraction of the encapsulated active substances is generallyobserved after administration.

On the basis of these discoveries, the instant invention aims atproviding new composition of particles of nanometric or micrometricdimensions, which allows easy encapsulation of chemical compounds, andespecially of active substances.

Another object of the invention is to provide compositions comprisingparticles having no traces of organic solvent and surfactants, which mayadvantageously be substituted to the particles known from the prior artin the vectorisation of active substances. Within this context, anobject of the invention is especially to provide compositions which makeit possible to achieve a controlled release of active substances.

Another object of the invention is to provide compositions intended toadminister active substances by ensuring “surreptitious (furtive)activity” in the organism, that is to say a capacity to circulate in aprolonged manner in the organism, particularly in the blood system,avoiding recognition by the immune system.

It is also an object of the invention to provide dispersions ofparticles free from surfactants and having nevertheless sufficientstability to be stored for a duration of about at least several days.

Thus, according to a first aspect, the present invention relates to acomposition comprising an aqueous dispersion of particles (p) of meanhydrodynamic diameter between 50 and 5000 nm, wherein said particlescontain in association:

(A) polymers based on cyclodextrin units, with an average content of atleast 4 cyclodextrin units within their structure; and

-   -   (B) macromolecules of polysaccharides comprising groups G        capable of forming inclusion complexes with the cyclodextrins        present in the structure of the said polymers (A), with an        average number of groups G per polysaccharide macromolecule at        least equal to 3,    -   wherein said compounds (A) and (B) are water-soluble in the        isolated state, i.e. preferably having a solubility at least        equal to 1 g/l, and advantageously at least equal to 3 g/l, at        25° C.

The term “Composition comprising an aqueous dispersion of particles” isunderstood, within the context of the present invention, to mean acomposition containing an aqueous medium (for example water,physiological serum or, more generally, an aqueous solution comprisingone or several solutes (especially one or several salt(s) and/orsugar(s) such as sucrose or glucose for example) at concentrationspreferably less than or equal to 20 g/l; or a mixture of thewater/alcohol type), wherein said particles are dispersed, theseparticles preferably being individual and advantageously having a rateof agglomeration below 80%, preferably below 50%, and preferably at mostequal to 10% by number. A composition according to the inventionadvantageously essentially consists in a dispersion of the particles (p)in water, in an aqueous solution of glucose (in particular a glucosesolution at a concentration of the order of 5% by mass), or even in aninjectable solution, at physiological pH, e.g. in physiological serum.However, a composition according to the invention can also be a morecomplex composition, for example a composition such as a single ormultiple emulsion, where the aqueous dispersion of the particlesaccording to the invention acts as a continuous phase or a dispersephase. A composition according to the invention can also comprise, inaddition to the particles based on the association of polymers (A) and(B), other particles of a mineral or organic nature.

The term “mean hydrodynamic diameter”, in the sense of the presentdescription, designates the size of a particle within an aqueous medium,which takes into account the average diameter of the particle in itsconformation in the aqueous medium, as well as its possible solvationlayer. The mean hydrodynamic diameter of a population of particleswithin an aqueous medium may especially be determined by quasi-elasticscattering of light within the medium under consideration, particularlyby an apparatus of the Nanosizer type. This type of apparatus alsoallows to determine for the population of particles a polydispersityindex of the hydrodynamic diameter, which indicates the distribution ofthe hydrodynamic diameters which is more or less narrow around the meanvalue.

Preferably within a composition according to the invention the meanhydrodynamic diameter of the particles (p) is greater than or equal to80 nm, and it is advantageously at least equal to 100 nm. The particles(p) may have, for certain applications, a mean hydrodynamic diameter ofbetween 1 and 5 microns (preferably less than 3 microns, if need be),but it is generally preferred for the mean hydrodynamic diameter of theparticles (p) to be less than or equal to 500 nm. More preferably, themean hydrodynamic diameter of the particles (p) is less than or equal to400 nm, and advantageously less than or equal to 300 nm. Thus, in aparticularly advantageous manner, the mean hydrodynamic diameter of theparticles (p) is between 100 and 300 nm, and even more advantageouslybetween 150 and 250 nm. Moreover, in a composition according to theinvention the particles (p) are generally of substantially sphericalmorphology, and the index of polydispersity of the hydrodynamic diameterthereof, as measured with the aid of an apparatus of the Nanosizer type,is generally between 0.1 and 0.3, and it is advantageously less than0.2.

The compounds (A) and (B) present in association within particlesaccording to the invention are compounds which form, in an aqueousmedium, an associative system (A+B) of the type of associations allpreviously defined, namely an associative system (A+B) of a type capableof leading, under certain concentration conditions, to an associativephase separation.

Thus the useful compounds (A) and (B) according to the invention are, ingeneral, compounds such that a mixture of two aqueous solutions of thesecompounds at a concentration of 50 g/l in a proportion ranging from 1:5to 5:1 by volume leads to the formation of a two-phase system of type(II) as defined previously.

The skilled person can easily check that candidate compounds (A) and (B)are actually capable of forming such a two-phase system by simplemixture of the aqueous solutions at ambient temperature. In order toselect a candidate of type (A) it is preferable to mix a solution ofthis compound with a solution of a compound (B) as described below inthe examples and, likewise, for a candidate of type (B) to make themixture with a solution of a compound (A) as described below in theexamples. In general, potential candidates can be tested withcomplementary compounds which have demonstrated their capacity forforming a system of type (II).

Preferably the compounds (A) and (B) present in a composition accordingto the invention are compounds such that a mixture of two aqueoussolutions of these compounds at concentrations ranging from 1 g/l to 10g/l, in a proportion ranging from 1:9 to 9:1 by volume, leads to theformation of particles of mean hydrodynamic diameter greater than 50 nmand less than 5000 nm.

These mixtures are extremely easy to produce, and it is sufficient thento check, by one of the conventional methods of analysis mentioned here,the appearance of the particles according to the invention.

The aqueous dispersion of particles (p) present within a compositionaccording to the invention characteristically constitutes a metastablesystem. Consequently, if a composition according to the invention issubjected to a sufficient shear stress (typically centrifugation at arate of at least 3000 r.p.m. for at least 20 minutes), an irreversibleevolution of the dispersion towards a system of type (II) as definedpreviously is generally observed. Nevertheless, it should be noted that,despite its metastable character, the aqueous dispersion of particles(p) present within a composition according to the invention generallyhas substantial stability.

The stability of the particles (p) in aqueous dispersion can especiallybe demonstrated by the retention of the mean hydrodynamic diameter ofthe particles (p) following storage. Thus, in general, after a storageof 24 hours, the mean hydrodynamic diameter of the particles (p) of acomposition according to the invention remains less than or equal to5000 nm. In most cases, following storage for 24 hours, the meanhydrodynamic diameter of the particles (p) of a composition according tothe invention remains less than or equal to 400 nm, preferably less thanor equal to 350 nm, and even more advantageously less than or equal to200 nm. Preferably the mean hydrodynamic diameter of the particles (p)remains less than or equal to 400 mu, and preferably less than or equalto 300 nm, following storage for 2 days, or indeed following storage for5 days, and even in certain cases after storage for 15 days. However, itshould be emphasised that in order to observe good retention of the meanhydrodynamic diameter of the particles (p) following prolonged storageit may sometimes be necessary to keep the composition in annon-oxidising atmosphere (for example under argon) so as to avoid ageingof the compounds (A) and/or (B), which is likely to call into questionthe stability of the metastable system formed.

The polymers (A) based on cyclodextrin units are essential constituentsof the particles (p) present in dispersion in the composition accordingto the invention. These polymers (A) include on average at least 4cyclodextrin units, preferably at least 9 cyclodextrin units, andadvantageously at least 15 cyclodextrin units. In a particularlyadvantageous manner, it is preferable for the polymers (A) to compriseon average at least 100 cyclodextrin units, and advantageously at least200 cyclodextrin units. Typically, the polymers (A) comprise on averageat least 400 cyclodextrin units. The average number of cyclodextrinunits present in the polymers (A) of a composition according to theinvention may be example be established by steric exclusionchromatography and by nuclear magnetic resonance.

The cyclodextrin units present within the polymers (A) can, in general,be α-cyclodextrins, β-cyclodextrins, γ-cyclodextrins, or mixtures of atleast two of these types of cyclodextrins. Nevertheless, thecyclodextrin units present in the polymers (A) preferably compriseβ-cyclodextrins. In a particularly advantageous manner, all thecyclodextrin units present in the polymers (A) are β-cyclodextrins.According to a particular variant, the cyclodextrin units present in thepolymers (A) are a mixture of β-cyclodextrins and α-cyclodextrins.According to this variant, it is generally preferred that theα-cyclodextrins represent less than 50%, and preferably less than 30% ofthe cyclodextrins present.

Within the polymers (A), the cyclodextrin units are generally linked toone another by hydrocarbon chains, linear or branched, which may beinterrupted by one or several oxygen atoms, and as these chains arepreferably alkyl, alkenyl or alkynyl chains, or also polyether chains,these chains may be substituted by hydrophilic groups (hydroxy groupsfor example). The chains linking the cyclodextrin units to one anotherhave at least 3 carbon atoms and preferably from 4 to 50 carbon atoms,the shortest path between two cyclodextrin units being preferablyconstituted by a chain having between 3 and 8 carbon atoms.Advantageously the hydrocarbon chains linking two cyclodextrin units toone another within a polymer (A) satisfy the general formula, a group offormula —O—(—CH₂—CHOR^((n))—CH₂)—O—, where n is an integer between 1 and50 (generally between 2 and 10) and where, in each of the n units(—CH₂—CHOR—CH₂), R^((n)) designates either a hydrogen atom or a—CH₂—CHOH—CH₂—O— chain linked to a cyclodextrin unit of the polymer.

Thus, the polymers (A) may typically be obtained by a polycondensationof cyclodextrin and epichlorhydrin molecules in a basic medium(generally in an aqueous medium with soda added, at a concentration bymass of 10 to 40%), the molar ratio cyclodextrins/epichlorhydrin beingpreferably between 1:15 and 1:1, and advantageously between 1:15 and1:8. For further details concerning this synthesis and control of themean number of cyclodextrin units integrated within the polymers (A)obtained according to this process, reference may be made in particularto the article by E. Renard et al, published in the European PolymerJournal, vol. 33, No 1, pp 49-57 (1997).

Regardless of the exact nature of the hydrocarbon chains linking thecyclodextrin units to one another, it is generally preferred that thetotal mass of the cyclodextrin units present within the polymers (A)represent at least 30%, advantageously at least 40%, and even morepreferably at least 50%, of the total mass of the polymers (A), thistotal mass of the cyclodextrin units generally representing between 30and 80%, and preferably between 40 and 60% of the total mass of thepolymers (A).

Moreover the polymers (A) present in a composition generally have a meanmolar mass by number of between 10 000 and 3 000 000 g/mole,advantageously between 20 000 and 2 000 000, and preferably between 100000 and 1 500 000 g/mole. The polymers (A) preferably have the lowestpossible polydispersity index (that is to say a ratio of the mean molarmass by weight to the mean molar mass by number), preferably less than3, and even more advantageously less than 2.

The polysaccharide molecules (B) present in the particles of thecompositions according to the invention include in a specific mannergroups G which are capable of forming inclusion complexes with thecyclodextrin units of the polymers (A).

These groups G can in particular be linear or branched aliphatic groupshaving 8 to 18 carbon atoms. Advantageously they are linear alkyl groupshaving 8 to 18 carbon atoms. According to a particular variant, they mayalso be adamantyl groups.

Thus the macromolecules (B) present in the composition according to theinvention are polysaccharides grafted by groups G as defined above.Preferably the groups G are aliphatic groups, and advantageously linearalkyl groups having 8 to 18 carbon atoms, or adamantyl groups, thesegroups being generally linked to the polysaccharide by way of an ester—COO-bond.

In a preferably manner, the grafted polysaccharides (B) are substitutedpolysaccharide derivatives, selected from amongst dextran, chitosan,amilose, amilopectin, hyaluronic acid, cellulose derivatives, starch,pullulan, pectin, alginates, heparin, carragheenans, fucan, curdlan,xylan, polyguluronic acid, xanthan, arabinan, polymannuronic acid, andtheir derivatives (such as for example dextran sulphate, amilose esters,or even cellulose acetate), these polysaccharides generally having amean molar mass by mass between 5 000 and 2 000 000 g/mol, andpreferably between 6 000 and 70 000. Advantageously, the macromolecules(B) are dextrans grafted preferably by linear alkyl groups comprising 8to 18 carbon atoms or by adamantyl groups.

Substituted polysaccharides which are useful as molecules (B) accordingto the invention may be obtained for example by reaction of apolysaccharide and an acyl chloride ROCl (R designating an aliphaticchain as defined above) in proportions corresponding to the mean amountof aliphatic chains R which it wished to graft onto the polysaccharide.Preferably in this case the reaction of the polysaccharide of the acylchloride ROC1 takes place at a temperature of between 30 and 90° C.(typically at 80° C.) and advantageously in the presence of a base suchas an amine, particularly of the pyridine type. An example of synthesisof this type is in particular described in an article by F. Arranz et alpublished in Polymer, vol. 29, pp 507-511 (1988).

The exact number and nature of the groups G present on themacromolecules (B) are preferably adapted to the nature of the polymer(A) used. In this connection it should in fact be emphasised that thecompounds (A) and (B) must be chosen from amongst the associativesystems which may, under certain concentration conditions, lead to anassociative phase separation.

Thus when the polymer (A) comprises α-cyclodextrin units, it isgenerally preferable that the groups G present on the macromolecules (B)should be alkyl groups, preferably linear, having 6 to 18 carbon atoms,and preferably 8 to 10 carbon atoms.

When the polymer (A) comprises β-cyclodextrins, it is most oftenadvantageous that the groups G present on the macromolecules (B) arealkyl groups, preferably linear, having 10 to 18 (preferably 12 to 16)carbon atoms, or adamantyl groups. If the polymer (A) contains onlycyclodextrin units of the β-cyclodextrin type, it is particularlyadvantageous that the groups G are linear alkyls having 10 to 16(preferably 12 to 16) carbon atoms, or adamantyl groups, these groupsthen preferably being linear alkyls having 12 carbon atoms or adamantylgroups.

In the case where the polymer (A) comprise γ-cyclodextrin units, it isgenerally preferable that the groups G present on the macromolecules (B)are alkyl groups, preferably linear, having 12 to 16 carbon atoms.

Moreover, the average number of groups G present on the macromolecules(B) is, characteristically, at least equal to 3 chains permacromolecule. As a general rule, this average number of groups G is allthe greater as the size of the macromolecule is high and the number ofcyclodextrin units in the compound (A) is substantial. It is most oftenadvantageous that the average number of groups G present on themacromolecules (B) is at least equal to 5, and advantageously at leastequal to 8. In general, the average number of groups G present on themacromolecules (B) remains less than or equal to 50, and advantageouslyless than or equal to 15.

As regards the grafted polysaccharide macromolecules, a “rate ofgrafting by the groups G” can be determined reflecting the quantity ofgroups G related to the size of the polysaccharide chain, that is to sayto the number of saccharide units of the polysaccharide chain. This rateof grafting by the groups G is calculated on the basis of a NMR spectrumof the proton of the grafted polysaccharides, and it corresponds to theratio of the integrations of the protons of the groups G, relative tothe integrations of the protons of the polysaccharide skeleton. Thisrate is calculated according to the following formula:rate of grafting by groups G (%)=(integration of the signalcorresponding to the protons of the groups G/number of protons on thegroups G)/integration of the signal corresponding to the protons of thepolysaccharide skeletons/number of protons on the polysaccharideskeletons)

The rate of grafting by the groups G of grafted polysaccharidemacromolecules (B) is in general equal to 1%, and preferably at leastequal to 2%. Advantageously this rate is greater than or equal to 3%.

However, in the more general case the number of groups G present on themacromolecules (B), most often hydrophobic, is limited by the fact thatthe compounds (B) must, characteristically, be water-soluble. It isequally desirable, as a general rule, to limit the number of groups Gpresent on the macromolecules (B) in such a way as to avoid phenomena ofauto-association between the macromolecules (B), more particularly whenthe groups G are hydrophobic groups of the aliphatic chain type. Becauseof this, the rate of grafting by the groups G is generally less than orequal to 8%, and preferably less than or equal to 6%. Thus it istypically between 3 and 4%.

In general, it is preferred that the macromolecules (B) have a meanmolar mass by weight at least equal to 20 000 g/mole, preferably between20 000 and 100 000 g/mole. It is preferred that the polymers (A) have apolydispersity index (that is to say a ratio of the mean molar mass byweight to the mean molar mass by number) as low as possible, preferablyless than 3, and even more advantageously less than 2.

In a particularly preferred manner the compounds (A) and (B) presentwithin the particles (p) of a composition according to the invention canbe chosen from amongst the following associations:

-   -   Family 1: polymers (A) having from 18 to 1000, preferably from        100 to 600 β-cyclodextrin units/polysaccharides (B) of molecular        mass between 6 000 and 70 000 (preferably dextrans) grafted by        C12 aliphatic groups (preferably linear alkyl chains) and having        a hydrophobic substitution rate of 3 to 5%, preferably less than        4%.    -   Family 2: polymers (A) having from 100 to 600, preferably from        200 to 500 β-cyclodextrin units/polysaccharides (B) of molecular        mass between 6 000 and 70 000 (preferably dextrans) grafted by        C10 aliphatic groups (preferably linear alkyl chains) and having        a hydrophobic substitution rate of 5 to 7%.    -   Family 3: polymers (A) having from 18 to 1000, preferably from        100 to 600 β-cyclodextrin units/polysaccharides (B) of molecular        mass between 6 000 and 70 000 (preferably dextrans) grafted by        adamantyl groups and having a hydrophobic substitution rate of 3        to 4%.

Regardless of the exact nature of the compounds (A) and (B) used, it isgenerally preferred that within the particles (p) of a compositionaccording to the invention the molar ratio of the total quantity ofcyclodextrin units present within the polymers (A) relative to the totalquantity of groups G present in the polysaccharide macromolecules (B) isbetween 1:3 and 3:1. This ratio is preferably at least equal to 0.7, andadvantageously at least equal to 0.8. Furthermore, it is preferred thatthis ratio is less than or equal to 1.5, and advantageously less than orequal to 1.3. Typically this ratio may for example be between 0.9 and1.1.

As a general rule, the concentration of compounds (A) and (B) within acomposition according to the invention can vary to quite a large extent.However, most often the concentration of polymers (A) within the aqueousdispersion present in the composition according to the invention isbetween 0.01 and 10 g/l, advantageously between 0.02 and 2 g/l, and evenmore advantageously between 0.2 and 2 g/l. With regard to theconcentration of macromolecules (B) within the aqueous dispersion, thisis generally between 0.01 and 10 g/l, preferably between 0.08 and 9 g/l,and even more preferably between 0.8 and 8 g/l. Furthermore, it isgenerally preferred that the total concentration of polymers (A) and ofmodified macromolecules (B) within the aqueous dispersion is between0.01 and 20 g/l.

In most cases, within a composition according to the invention themajority of the polymers (A) and of the macromolecules (B) is localisedwithin the particles (p). Thus in general at least 80% by mass(preferably at least 85% by mass, and advantageously at least 90% bymass of the compounds (A) and (B) present in a composition according tothe invention are contained in the particles (p).

According to another aspect, the present invention also relates to amethod of preparing a composition as described previously. This methodcomprises a step (E), extremely simple to implement, which consists ofeffecting a mixture of an aqueous solution (S_(A)) comprising polymers(A) as defined previously and an aqueous solution (S_(B)) comprisingpolysaccharide macromolecules (B) as defined above, by choosing thevolumes and the concentrations of the said solutions (S_(A)) and (S_(B))in such a way as to obtain, after the mixing, an aqueous medium wherethe respective concentrations C_(A) and C_(B) in the said compounds (A)and (B) belong to the range for formation of a metastable dispersion forthe auto-associative system (A+B) used.

Within the context of the present invention, the “range for formation ofa metastable dispersion of an auto-associative system (A+B)” isunderstood to mean the range of concentrations of compounds (A) and (B)the existence of which was discovered by the inventors for theassociations of type all, in which a direct mixture of compounds (A) and(B) as defined previously leads to the formation of a dispersion ofparticles of dimensions between 50 and 5000 nm. The term “range ofconcentrations” as used here designates a set of pairs (C_(A); C_(B)).This term “range” refers to the ternary water/compound(A)/compound(B)diagrams which can be established for a system (A+B), by observing thesystem formed for the different water contents of water, compound (A)and compound (B). On such a ternary diagram the set of pairs (C_(A);C_(B)) in which it is necessary to choose the respective concentrationsof compounds (A) and (B) in the final medium in order to observe theformation of the metastable dispersion appears in effect in the form ofa continuous zone, known as the “range of concentrations”. In so far asthe establishment of ternary diagrams for systems of type (A+B) isconcerned, reference may be made in particular to the article“Macromolecular assemblies generated by inclusion complexes betweenamphiphatic polymers and b-cyclodextrin polymers in aqueous media” (C.Amiel et al, ACS: Symposium Series, No. 780, edited by C. McCormik“Stimuli-Responsive Water-Soluble and Amphiphatic Polymers”, chapter 4(2000).

In general, on a ternary water/compound(A)/compound(B) diagram of theaforementioned type, the range of formation of the metastable dispersionwhich has been discovered by the inventors is situated at the boundarybetween the ranges of formation of the two thermodynamically stablesystems known from the prior art, namely the aforementioned systems oftype (I) and (II).

In most cases, a composition according to the invention can be obtainedfrom the majority of the associative systems of type (A+B) as definedpreviously if the medium obtained at the end of step (E) verifies thefollowing two conditions:

-   -   the sum of the concentrations C_(A)+C_(B) is between 0.1 and 10        g/l, and preferably between 1 g/l and 10 g/l; and    -   the molar ratio of the total quantity of cyclodextrin units        present within the polymers (A) introduced, relative to the        total quantity of aliphatic chains present as substituents on        the polysaccharide macromolecules (B) introduced, is between 1.3        and 3.1, and preferably between 1.2 and 2.1.

The solution (S_(A)) used in step (E) generally has a concentrationbetween 0.01 g/l and 20 g/l, this concentration being advantageouslybetween 0.1 and 10 g/l. The solution (S_(B)) for its part most often hasa concentration between 0.01 g/l and 20 g/l, this concentrationpreferably being between 0.1 and 10 g/l.

As a general rule, and especially when the solutions (S_(A)) and (S_(B))have the aforementioned concentrations, the ratio of the total volume ofsolution (S_(A)) introduced to the total volume of solution (S_(B))introduced is most often between 1:9 and 9:1, and preferably this ratioby volume is between 1:5 and 5:1. In particular when the macromolecules(B) are grafted polysaccharides, of the modified dextran type, it ispreferred that this ratio is between 1:5 and 1:1.25 and preferablybetween 1:3 and 1:0.75.

As a general rule, the method according to the present invention isextremely simple to implement. Thus, step (E) of the method according tothe invention consists of a simple mixture, which is generally carriedout at ambient temperature, that is to say most often between 15° C. and30° C. Moreover, it should be noted that the method of preparationaccording to the invention can be limited merely to carrying out step(E).

Especially so as to produce the most homogeneous possible mixture of thesolutions (S_(A)) and (S_(B)), it is often advantageous to carry outstep (E) with agitation, but most of the time this agitation is notnecessary in order to observe the formation of the metastabledispersion.

In a more general manner, the inventors have evidenced that, subject tobeing within the adapted range of concentrations and producing a directmixture as quickly as possible of the solutions (S_(A)) and (S_(B)), themixture of the said solutions leads spontaneously to the formation ofthe metastable dispersion.

In this connection, it should be emphasised that the method according tothe invention does not necessitate the use of solvents or surfactants.For this reason a composition according to the invention is generallyfree from any trace of organic solvent or surfactant. Thus, according toa particular embodiment a composition according to the invention maycomprise water and compounds (A) and (B) as defined previously, to theexclusion of any other compound.

Nevertheless, taking account of the presence of cyclodextrin unitswithin their structure, the particles (p) present within a compositionaccording to the invention are particularly adapted to produce theencapsulation of chemical compounds, and quite particularly to producethe encapsulation of chemical compounds having groups of a hydrophobicnature. The particular use of the compositions according to theinvention for this purpose constitutes a particular object of thepresent invention.

The particles (p) present in the compositions according to the inventionare able to integrate, in encapsulated form, numerous types of neutralor charged chemical compounds. The compounds capable of beingencapsulated within the particles (p) of a composition according to theinvention comprise in particular the compounds having hydrophobicgroups, particularly alkyl groups, having in general 6 to 128 carbonatoms.

More specifically, the particles (p) present in a composition accordingto the invention are particularly well adapted to produce encapsulationof compounds capable of forming inclusion complexes with thecyclodextrin units which they comprise. For further details with regardto the formation of inclusion complexes between chemical compounds andcyclodextrins, as well as with regard to the nature of the compoundscapable of forming such complexes, reference may be made to“Cyclodextrins and their inclusion complexes”, Szejtli J., AcademiaKiado, Budapest, 1982.

In general, simply placing the aforementioned chemical compounds incontact with a composition according to the invention is sufficient inorder to produce the encapsulation of chemical compounds by theparticles (p), in particular when the said composition is essentiallybased on water and particles (A) and (B).

In this scope, a composition according to the invention may for examplebe used as an absorbent composition, particularly for entrapping toxicagents or pollutants present in an aqueous medium, and in particular toeliminate compounds of the hydrocarbon type (particularly polycyclicaromatic hydrocarbons), halogenated aromatic compounds (such aschlorobenzene or chlorophenols), phthalic esters, or also iodine,particularly radioactive iodine, or pollutants of the pesticide type ortextile dyes. In order to do this, it is generally sufficient tointroduce the composition based on particles (p) within the medium to bepurified. The particles (p) can, in this type of application, absorb(encapsulate) a quantity of compounds to be eliminated at least equal to0.1 mole of compound per mole of cyclodextrins included in theparticles, and may be up to 1 mole per mole of cyclodextrin, even up to2 mole per mole of cyclodextrin, particularly for pollutants of thehydrocarbon type, particularly of the polycyclic aromatic hydrocarbontype.

The compositions according to the invention having chemical compoundsencapsulated within their particles (p) may also be of interest inthemselves. According to a particular aspect, the present invention alsorelates to such compositions according to the invention, where theparticles (p) comprises at least one chemical compound (C), other thancompounds (A) and (B).

In this type of composition, said compound (C) is in general a compoundhaving groups of a hydrophobic nature, advantageously groups of the typehaving hydrocarbon chains having 8 to 18 carbon atoms, and preferably 10to 18 carbon atoms. In an advantageous manner, this compound (C) is acompound capable of forming an inclusion complex with one of thecyclodextrin units contained in the polymers (A) present in theparticles (p).

It is preferred that in a composition comprising a compound (C) of theaforementioned type the quantity of compound (C) integrated within theparticles (p) represents at least 0.5% by mass relative to the totalmass of the said particles (p). It is often advantageous in such acomposition that the ratio by mass (C)/(A+B) of the total mass of thecompounds (C) relative to the total mass of the compounds (A) and (B) isbetween 1% and 50%, this ratio being preferably greater than 2% andadvantageously greater than 3%.

As already emphasised in the instant description, a compositionaccording to the invention additionally comprising a compound (C) asdefined above can in particular be obtained by simply placing the saidcompound (C) in contact with a composition according to the inventionbased on a preformed aqueous dispersion of particles (p) based on thecompounds (A) and (B). In this context, it is naturally preferable thatthe particles (p) are essentially based on compounds (A) and (B), to theexclusion of the presence of any other compound, in particular of acompound capable of forming an inclusion complex with the cyclodextrinunits present in the polymers (A). However, it is possible to envisageseveral successive steps for placing in contact with different types ofcompounds (C). The total quantity of compound (C) placed in contact withthe composition in the course of this or these steps preferablyrepresents between 4 and 100% by mass, and preferably between 10 and 80%by mass relative to the total mass of the particles (p) presentinitially in the composition. This quantity of compound (C) placed incontact with the composition advantageously represents between 10 and70% by mass, and more preferably between 20 and 50% by mass relative tothe total mass of the polymers (A).

In an advantageous manner, especially when it is desired to prepare acomposition according to the invention additionally comprising compounds(C) capable of forming an inclusion complex with one of the cyclodextrinunits contained in the polymers (A), the incorporation of the compounds(C) within the particles (p) may be achieved by using in the method ofpreparation according to the invention a solution (SA) comprising inaddition to the polymers (A) the compounds (C) which it is desired tointegrate into the particles (p), this compound (C) advantageouslyforming inclusion complexes with cyclodextrin units present in thepolymers (A). In this context, within the solution (S_(A)) the ratio bymass of compound (C)/polymer (A) is preferably between 10 and 100%, andmore preferably between 20 and 50%. The presence of the compound (C)within the solution (S_(A)) does not generally modify the range offormation of the metastable dispersion for the auto-associative system(A+B) relative to the conditions to be implemented in the absence of thecompound (C). For this reason, in the presence or absence of thecompound (C) the volumes and the concentrations of the solutions (S_(A))and (S_(B)) are chosen in such a way as to obtain, following themixture, an aqueous medium where the respective concentrations C_(A) andC_(B) in the said compounds (A) and (B) belong to the range of formationof a metastable dispersion for the auto-associative system (A+B).

The exact nature of the compound (C) which may be encapsulated in acomposition according to one of the aforementioned methods, can vary toquite a large extent. However, particularly in so far as the polymers(A) and the macromolecules (B) can be chosen from amongst non-toxic andbiocompatible compounds and the presence of traces of organic solventsor surfactants can be avoided, one of the principal applications whichcan be envisaged for a composition according to the invention is thevectorisation of active substances, particularly of compounds having atherapeutic or cosmetic effect.

Thus, according to a particularly advantageous embodiment, a compositionaccording to the invention may comprise by way of compound (C) withinits particles (p) at least one active compound by way of medicament,this active compound (C) by way of medicament preferably being capableof forming an inclusion complex with one of the cyclodextrin unitscontained within the particles (p).

Such a composition according to the invention can be used in general byway of a pharmaceutical composition for administration by injection ororally, or also by the dermal or subcutaneous route, nasally, by thepulmonary route or by the ocular route and, more broadly, for anyadministration at the level of a mucous membrane, or at the level of aprecise site (tumour, lumen of certain blood vessels, . . . ). In thiscontext it is most often preferable that the composition is essentiallyconstituted by water and compounds (A), (B) and (C), possibly inassociation with one or several pharmacologically acceptable excipientsand adapted to the administration route envisaged. However, broadly thecomposition according to the invention can, in this type of application,take the form of any pharmaceutical formulation integrating an aqueousdispersion of the particles (p) comprising the active compound (C) byway of medicament. In the case of a composition specifically intendedfor administration by intravenous injection, it is generally preferredthat the particles (p) integrating the compound (C) have a meanhydrodynamic diameter at least equal to 200 nm. With regard to thecompositions intended for administration by intramuscular injection, itis preferred that the particles (p) having the compound (C) integratedin them have a mean hydrodynamic diameter between 200 and 5000 nm,preferably less than 1000 nm.

The compositions according to the invention, which have particles (p)based on grafted polysaccharides integrated in them by way of compounds(B), are of particular interest in terms of bioadhesion, which makesthem extremely advantageous for application to the mucous membranes. Foran application by the ocular route, it has proved particularlyinteresting that the compounds (B) present in the particles (p) aregrafted hyaluronic acids.

The compositions according to the invention comprising an activecompound (C) by way of medicament within their particles (p) generallyinduce, following their administration, a progressive release of theencapsulated compound (C), especially when the said compound (C) is acompound capable of forming an inclusion complex with one of thecyclodextrin units comprised in the particles (p), in particular whenthe said composition is administered in a patient intravenously. Thuswith the aid of such a composition it is possible to achieve theprolonged administration of the active compound (C), in particular whenthis compound is chosen from amongst tamoxifen or its derivatives, orfrom amongst piroxicam and its derivatives. Numerous active substancesare capable of being encapsulated in the particles (p) of thecompositions according to the invention with the aim of prolongedsalting out, and particular of the antiinfectious, anti-inflammatory,antibacterial and antiparasitic agents, opioids, enzymes, or alsopolypeptides. Thus as active compounds (C) acting as a medicament whichcan be encapsulated in the particles (p), mention may be made inparticular of molsidomine, ketoconazole, gliclazide, diclofenac,levonorgestrel, paclitaxel, hydrocortisone, pancratistatin, ketoprofen,diazepam, ibuprofen, nifedipin, testosterone, tamoxifen, furosemide,tolbutamide, chloramphenicol, benzodiazepines, naproxene, dexamethasone,diflunisal, anadamide, pilocarpine, daunorubicin, doxorubicin anddiazepam.

In this context, without wishing to be associated in any way with aparticular theory, it seems that it could be suggested that the releaseof the compound (C) is effected with a balance of distribution betweenthe particles (p) and the external medium. Thus it seems that theinternal hydrophobic cavity of each cyclodextrin constitutes a potentialacceptor site for molecules of active substances or for lipophilicfragments thereof. The greater the affinity of the active substance forthe cyclodextrins, the more its release will be slowed down. It has beenpossible in particular to demonstrate this concept with a modelmolecule, benzophenone, having a high affinity for cyclodextrins.Because of this, an active compound (C) acting as a medicament containedin the particles (p) of a composition according to the invention is mostoften released in a preferred manner at the level of the cells or thetissues where this compound is consumed, that is to say most often whereit plays an effective therapeutic role.

As regards the compositions according to the invention comprising anactive compound (C) acting as a medicament, it should be noted that theparticles (p) can in general be administered as such by the oral route.In this case they can make it possible to achieve the oraladministration of compound (C) with an unpleasant taste or smell (theencapsulation is generally capable of masking this taste or this smell)or of a compound (C) which is fragile and/or difficult to absorb orally,such as for example a compound chosen from amongst theantiinflammatories such as piroxicam, ibuprofen and ketoprofen,hypoglycaemic agents such as glicazide, contraceptive agents such asD-norgestrel, or also antifungal or antiparasitic agents such asketoconazole or albendazole.

Moreover, without wishing to be associated in any way with a particulartheory, it seems that it could be suggested that in so far as thecompounds (B) are polysaccharides carrying groups G, the structure ofthe particles (p) is as a general rule such that the external layer ofthe particles (p) is essentially constituted by polysaccharides. In anycase, in particular when the polysaccharides (B) are dextrans, theparticles (p) generally have a tendency to adhere to the surface ofcertain mucous membranes, at the level of which they then deliver theactive substance (C) which they contain, generally in a progressivemanner. Thus by applying a composition according to the inventioncomprising a compound (C) with a therapeutic effect to a given (nasal,ocular, . . . ) mucous membrane it is possible to achieve a selectiveadministration of the compound at the level of this mucous membrane.Moreover, it also seems that it could be suggested that, during oraladministration, the specific structure of the particles (p) favourstheir translocation through the digestive epithelium, and their passagein the intact state into the blood system, where they can then allowprolonged release of the compound (C).

In the case where the particles (p) are intended for the vectorisationof compounds of the medicament type, it is generally advantageous thatthe compounds (A) and/or (B) are substituted by groups permittingcellular targeting. In this context it may be of interest for examplethat the compounds (A) and/or (B) are complexed by ligands of the folicacid type. The particles (p) then constitute specific so-called“third-generation” ligands.

Moreover, in such a way as to improve the “surreptitious” character ofthe particles (p) according to the invention (that is to say theircapacity to circulate in a prolonged manner in the organism whilstavoiding detection by the immune system), it may be advantageous thatthe particles (p) according to the invention have external groups of thepropylene glycol (PEG) type. In order to achieve this, use may be madeby way of compounds (B) of polysaccharide molecules (preferablydextrans) carrying not only groups G as defined previously but also PEGchains. Another embodiment of particles grafted by PEG groups consistsof adding to the system of compounds (A) and (B), in addition to thecompounds (C), compounds of the PEG-[Alk] type, where Alk represents aC₁₀ to C₁₈ alkyl group, preferably a C₁₂ to C₁₆ alkyl group, or anadmantyl group. If need be, as for the compounds (C), the addition ofthe compounds of the PEG-[Alk] type can be effected before or afterformation of the particles from the associative system (A+B). However,it is generally preferred that this addition is effected after formationof the particles.

However, as a general rule, the presence of PEG groups is not necessaryin order to ensure the surreptitious activity and, most often, theparticles (p) prove capable of ensuring for example the vectorisation ofthe compounds with a reduced plasma half-life or with high toxicity,particularly at the level of the mononuclear phagocytes.

According to another embodiment, the compounds (C) which may be presentwithin the particles (p) can also be cosmetic active substances, and thecomposition according to the invention can then be used advantageouslyby way of a cosmetic composition.

In this context the compounds (C) can for example be odorous compounds,for example of the terpene type, or a mixture of such compounds(perfumes, essences, . . . ). In such a composition the odorouscompounds generally have a lower irritating power than in thenon-encapsulated state, and they are released in a retarded manner,which improves the retention of the perfume. In the same way, othertypes of cosmetic agents preferably having a hydrophobic character maybe immobilised as compound (C) within the particles (p) of a compositionaccording to the invention, then released in a progressive manner. Thusa composition according to the invention may for example permit thecontrolled release of antiperspirant agents or also antibacterialagents. In a composition according to the invention intended forcosmetic use, the compound (C) may also be a colouring which is anirritant or has a certain toxicity, so that encapsulation thereof makesit possible to reduce the undesirable effects.

In a more general manner, the compositions according to the inventionwith the compounds (C) integrated in them can be used in order toachieve a progressive release of the said compounds (C) within a mediuminto which they are introduced or also in order to limit the contactbetween the compounds (C) and the said medium, for example with a viewto protection of the compounds, when they are molecules which arefragile in relation to the medium under consideration, or also in orderto isolate the compounds which may be pollutants (toxic agents,irritants, reagents, . . . ) for this medium. This general use of thecompositions according to the invention comprising compounds (C) withinthe particles (p) constitutes another object of the present invention.

Whether or not they comprise a compound (C) by way of an addition, thecompositions according to the invention can generally be subjected to alyophilisation step, particularly when the composition is essentiallyconstituted by an aqueous dispersion of the particles (p). If needed,this lyophilisation step is generally carried out by cooling thecomposition abruptly (generally in liquid air or liquid nitrogen), thensublimating the water under strong negative pressure. The compositionsobtained at the end of such a lyophilisation step, which are generallypresent in the form of a compact powder of fluffy appearance, and whichmay be dispersed in the water in order to lead to the reconstitution ofa dispersion of particles of the type of particles (p), constituteanother particular object of the present invention.

The characteristics and advantages of the present invention will becomeeven more apparent from the illustrative examples set out below.

The appended single drawing shows an enlarged view of particlesaccording to the invention.

EXAMPLES Example 1 Synthesis of Polymers (A) Based on β-CyclodextrinUnits

Two polymers P1 and P2 were prepared based on cyclodextrins by reactingβ-cyclodextrin (denoted by β-CD) with epichlorhydrin in a basic medium,using the mode of operation described in the European Polymer Journal,vol. 33, No. 1, pp 49-57 (1997).

1.1—Synthesis of Polymer P1

5 g of β-CD were dissolved in an aqueous solution of soda at 33% by massin a two-necked flask. This mixture was left under agitation at ambienttemperature (20° C.) for 24 hours in such a manner as to produce thedeprotonation of the hydroxyl groups.

Then 2.7 ml of epichlorhydrin were introduced into the medium (molarratio β-CD/epichlorhydrin=1/7) and the mixture was agitated vigorouslyand brought to 30° C. The medium was left under these conditions for 3hours.

The reaction was then stopped by addition of acetone which dissolves theexcess of epichlorhydrin. The supernatant acetone solution was theneliminated.

The polymer (precipitate) was dissolved in distilled water and thesolution was brought to pH 12 and agitated for 24 hours. The pH valuewas then brought to 7 (by the addition of hydrochloric acid 6N), thenthe mixture was ultrafiltered through a membrane with a cut-offthreshold of 1000 Dalton in order to eliminate the salts.

The polymer P1 obtained following these different steps was thenlyophilised then stored in the freezer.

1.2—Synthesis of Polymer P2

As for polymer P1, 5 g of β-CD were dissolved in an aqueous solution ofsoda at 33% by mass in a two-necked flask. This mixture was left underagitation at ambient temperature (20° C.) for 24 hours in such a manneras to produce the deprotonation of the hydroxyl groups.

Then 3.8 ml of epichlorhydrin were introduced into the medium (molarratio β-CD/epichlorhydrin=1/10) and the mixture was agitated vigorouslyand brought to 30° C. The medium was left to develop until it reachedthe immediate vicinity of gelation of the reaction medium, that is tosay until a medium with high viscosity was obtained.

The reaction was then stopped by addition of acetone which dissolves theexcess of epichlorhydrin. The supernatant acetone solution was theneliminated.

The polymer (precipitate) was dissolved in distilled water and thesolution was brought to pH 12 and agitated for 24 hours. The pH valuewas then brought to 7 (by the addition of hydrochloric acid 6N), thenthe mixture was ultrafiltered through a membrane with a cut-offthreshold of 1000 Dalton in order to eliminate the salts. A secondultrafiltration was then carried out through a membrane with a cut-offthreshold of 100 000 Dalton in such a way as to eliminate the fractionsof low molar mass.

The polymer P2 obtained following these different steps was thenlyophilised then stored in the freezer.

The characteristics of the polymers P1 and P2 are set out in the tablebelow (the molecular masses shown in this table are as measured by SECchromatography, with pullulan calibration, and the average number ofβ-cyclodextrin units per copolymer was calculated from this molecularmass). TABLE 1 characteristics of polymers P1 and P2 Average number ofβ- cyclodextrin units per Polymer (A) M (g/mol) copolymer P1   40 000 20P2 2 600 000 1350

Example 2 Synthesis of Dextrans Modified by Alkyl Chains (B)

Different dextrans modified by alkyl chains were prepared by reactingdextran and different acyl chlorides using the mode of operationdescribed in Polymer, Vol. 29, pp 507-511 (1988).

More precisely, dextran of a molecular mass equal to 40 000 (dextranT40) was reacted with different alkyl chlorides (nature and quantityspecified in Table 2 below) in the presence of pyridine (protondetector) and dimethylamino pyridine (DMAP) (catalyst).

In each of the syntheses effected, 4g of dextran were solubilised in 100ml of previously distilled dimethylformamide in the presence of 1 g oflithium chloride LiCl. The reaction was carried out by adding to themedium obtained 0.5 g of DMAP, 0.031 ml of pyridine and alkyl chloride,and leaving it to react at 80° C. for 3 hours.

The polymer obtained was the purified by precipitation in isopropanol,then by dialysis after solubilisation in water. During this step thepolymer was fractionated as a fuinction of its rate of modification bydifference in solubility in water.

For this purpose, after solubilisation in water the solution was pouredinto a test tube and it was left to rest for 4 hours. Following thisdecantation, three fractions (upper, median and lower) of equal volumeswere then separated. In each of the syntheses, one or several of thesefractions (as indicated in Table 2 below) were dialysed with pure waterthrough membranes with a cut-off threshold of 6000-8000 Dalton.Following the dialysis of the fraction under consideration, the modifieddextran obtained was characterised by NMR in order to determine the rateof substitution by the alkyl chains, then it was lyophilised.

The characteristics of the modified dextrans DM1-DM5 thus prepared areset out in Table 2 below. TABLE 2 characteristics of the modifieddextrans DM1-DM5 Modified Quantity of Fraction taken Rate dextran Natureof the alkyl chloride following of (B) grafted chains used decantationgrafting DM1 linear C₁₂ alkyl  0.5 ml upper fraction 5.8% chains DM2linear C₁₀ alkyl  0.4 ml median fraction + 5.3% chains lower fractionDM3 linear C₁₂ alkyl 0.43 ml upper fraction 4.2% chains DM4 linear C₁₂alkyl 0.43 ml median fraction 3.3% chains DM5 adamantyl 0.54 g upperfraction   3% groups

Example 3 Preparation of Aqueous Dispersions of Particles According tothe Invention

Different aqueous dispersions according to the invention were producedby mixing a volume V(A) of an aqueous solution of polymers (A) at aconcentration C(A) with a volume V(B) of an aqueous solution of modifieddextrans (B) at a concentration C(B). The conditions of the mixturesproduced are set out below: TABLE 3 conditions of mixtures Modi- Poly-fied Compo- mer dextran sition (A) (B) C(A) C(B) V(A) V(B) C1 P1 DM1  10mg/ml  10 mg/ml    1 ml    1 ml C2 P1 DM2  10 mg/ml  10 mg/ml    1 ml   1 ml C3 P2 DM3 0.6 mg/ml 0.6 mg/ml    1 ml    1 ml C4 P2 DM4   3mg/ml   3 mg/ml 0.375 ml 0.625 ml C5 P2 DM5 2.5 mg/ml 2.5 mg/ml    1 ml   1 ml

The mean diameters of the particles observed in the dispersions C1 to C4obtained immediately after the mixture and following 2 to 15 days ofstorage are set out in Table 4 below. TABLE 4 evolution of the meandiameters of the particles present in the compositions C1 to C4 Diameterof Diameter of Diameter of Diameter of particles particles particlesparticles Composition (t = 0) (t = 2 days) (t = 5 days) (t = 15 days) C1113 nm 167 nm 173 nm 228 nm C2 112 nm — 106 nm — C3 260 nm 310 nm — — C4130 nm 260 nm 270 nm 350 nm C5 240 nm⁽*⁾ — — —⁽*⁾At the end of 1 day of storage, the mean diameter of the particles ofthe composition C5 is 330 nm.

Example 4 Influence of the Molar Mass of the Polymer (A)

The mixture was produced with 1 ml of a solution of modified dextran DM3(10 mg/ml in water milliQ) with 1 ml of a solution of two polymers (A)of different molar masses (polymers P1 and P2 as defined in Example 1,of respective molecular masses 40 000 g/mole or 2 600 000 g/mole)equally at 10 mg/ml in water milliQ.

Table 5 below shows the evolution of the mean diameters of the particlesobtained in the two cases: TABLE 5 evolution of the mean diameters ofthe particles for two polymers (A) of different molar masses Diameter(nm) Polymer (A) t = 0 3 days 5 days 7 days 12 days P1 160 456 610gelation gelation M = 40 000 g/mole P2 198 230 261 255 275 M = 2 600 000g/mole

Thus it will be noted that the particles obtained with a polymer (A) oflow molecular mass are less stable than those obtained with a polymer(A) of higher molar mass (for P2 the diameter tends to stabilise after 5days).

In the case of P1, however, it should be noted that the evolutions ofdiameter are less significant when the concentrations of the startingpolymers are lower.

Example 5 Encapsulation of a Model Compound: Benzophenone

An aqueous solution of polymer P2 as defined in Example 1 (0.44 g/l) wasproduced, to which benzophenone was added with a molar ratio ofbenzophenone added: cyclodextrin equal to 1:1. This solution was leftunder agitation for 24 hours.

A composition of particles was prepared by mixture of 1 ml of thesolution thus obtained with 1 ml of a solution of modified dextran DM3as defined in Example 1 (0.44 g/l).

After mixture, the particles produced were ultracentrifuged (30 minutesat 40000 r.p.m. in a Beckman L7-55 centrifuge). The non-encapsulatedbenzophenone present in the supernatant was dosaged by spectrophotometry(absorption line at 261 nm).

The experiment was repeated using a molar ratio of benzophenone added:cyclodextrin equal to 1:3 for the solution of polymer P2.

The experiment was also repeated using modified dextran DM4 instead ofDM3.

Finally, the experiment was also repeated using a molar ratio ofbenzophenone added cyclodextrin equal to 1:3 for the solution of polymerP2 and also replacing the modified dextran DM3 by DM4.

In the 4 cases, the yields of encapsulation of the benzophenone arebetween 30 and 40% (less than 70% of the benzophenone initiallyintroduced is found in the supernatant).

With a molar ratio of benzophenone : cyclodextrin equal to 1:1 in theinitial solution of P2, particles are produced comprising 1% by mass ofbenzophenone. With a molar ratio of benzophenone: cyclodextrin equal to1:3, particles comprising 3% by mass of benzophenone are produced.

Example 6 Preparation of Compositions of Lyophilised Particles

Compositions of particles were prepared by mixture of two identicalvolumes (1 ml) of a solution of polymer (A) and a solution of modifieddextran (B) under the conditions set out in Table 6 below.

The receptacles containing them were immersed in liquid air. Theparticles were then lyophilised (Christ LDC-1) for 24 hours, with orwithout the presence of cryoprotector (saccharose) as appropriate. Itwas demonstrated in this context that the use of glucose and maltose isa cryoprotector poorly adapted to this type of particles.

The lyophilisates obtained were then mixed with water milliQ in such away as to reconstitute the compositions of particles.

The diameters before and after lyophilisation are set out in Table 6below. TABLE 6 tests of lyophilisation and demonstration of theredispersibility of the lyophilisates obtained Diameter (nm) Modifiedbefore Polymer dextran lyophili- after solution solution Cryoprotectorsation lyophilisation polymer P1 dextran DM3 no 273 ± 64 157 ± 52 0.5mol/l 0.5 mol/l polymer P2 dextran DM3 saccharose 130 ± 50 178 ± 64   1mol/l   1 mol/l polymer P2 dextran DM1 no 116 ± 40 117 ± 47   1 mol/l  1 mol/l

Example 7 Stability of the Compositions According to the Invention UnderShear

A composition of particles according to the invention was prepared bymixture of 5 ml of solution of polymer P2 at 10 mg/ml in water milliQand 5 ml of solution of modified dextran DM3 at 10 mg/ml in watermilliQ. The mean diameter of the particles obtained is 178 nm.

6.3 ml of the composition of particles produced was subjected to shearin Godel cone geometry (DG41) on a Rheo-Stress 100 flowmeter(Haake/Fisons). The study was carried out at 20° with a shear stressgoing from 0 to 2000 niPa in 300 seconds, maintained at 2000 mPa for 300seconds, then decreased from 2000 to 0 mPa in 300 seconds.

Following this treatment, the final diameter of the particles wasmeasured equal to 183 m. Thus, no significant evolution was noted.

1. A composition comprising an aqueous dispersion of particles (p) ofmean hydrodynamic diameter between 50 and 5000 nm, wherein saidparticles contain, in association: (A) polymers based on cyclodextrinunits, with an average content of at least 4 cyclodextrin units withintheir structure; and (B) macromolecules of polysaccharides comprisinggroups G capable of forming inclusion complexes with the cyclodextrinspresent in the structure of the said polymers (A), with an averagenumber of groups G per polysaccharide macromolecule at least equal to 3,wherein said compounds (A) and (B) are water-soluble in the isolatedstate.
 2. The composition of claim 1, wherein the particles (p) have amean hydrodynamic diameter greater than or equal to 80 nm and less thanor equal to 500 nm.
 3. The composition of claim 1 wherein the polymers(A) have on average at least 9 cyclodextrin units within theirstructure.
 4. The composition of claim 1 wherein the cyclodextrin unitspresent in the polymers (A) comprise β-cyclodextrins.
 5. The compositionof claim 1, wherein the polymers (A) are obtained by polycondensation ofcyclodextrin and epichlorhydrin molecules.
 6. The composition of claim 1wherein the polymers (A) have a mean molar mass by number of between 10000 and 3 000 000 g/mole.
 7. The composition of claim 1, wherein thegroups G are aliphatic groups, linear or branched, having 8 to 18 carbonatoms.
 8. The composition of claim 1, wherein the rate of grafting ofthe polysaccharides (B) by the groups G is between 1 and 8%.
 9. Thecomposition of claim 1, wherein the compounds (A) and (B) are chosenfrom amongst the following associations: polymers (A) having from 18 to1000 β-cyclodextrin units/polysaccharides (B) of molecular mass between6 000 and 70 000 grafted by C12 aliphatic groups and having ahydrophobic substitution rate of 3 to 5%; polymers (A) having from 100to 600 β-cyclodextrin units/polysaccharides (B) of molecular massbetween 6 000 and 70 000 grafted by Cl0 aliphatic groups and having ahydrophobic substitution rate of 5 to 7%; polymers (A) having from 18 to1000 β-cyclodextrin units/polysaccharides (B) of molecular mass between6 000 and 70 000 grafted by adamantyl groups and having a hydrophobicsubstitution rate of 3 to 4%.
 10. The composition of claim 1, whereinthe molar ratio of the total quantity of cyclodextrin units presentwithin the polymers (A) relative to the total quantity of aliphaticchains present by way of substituents on the polysaccharidemacromolecules (B) is between 1:3 and 3:1.
 11. The composition of claim1, wherein at least 80% by mass of the compounds (A and B) present inthe composition are contained in the particles (p).
 12. The compositionof claim 1, wherein in the particles (p) comprise at least oneadditional chemical compound (C) other than the compounds (A) and (B).13. The composition of claim 12, wherein the said compound (C) is acompound capable of forming an inclusion complex with one of thecyclodextrin units contained in the polymers (A) present in theparticles (p).
 14. The composition of claim 12 wherein the quantity ofcompound (C) integrated within the particles (p) represents at least0.5% by mass relative to the total mass of the said particles (p). 15.The composition of claim 12, wherein the compound (C) is a compoundhaving a therapeutic or cosmetic effect and that the said composition isa pharmaceutical or cosmetic composition.
 16. A method of preparation ofthe composition of claim 1, which comprises a step (E) which consists ofeffecting a mixture of an aqueous solution (S_(A)) comprising polymers(A) as defined in claim 1 and an aqueous solution (S_(B)) comprisingpolysaccharide macromolecules (B) as defined in claim 1, the volumes andthe concentrations of the said solutions (SA) and (SB) being chosen insuch a way as to obtain, after the mixing, an aqueous medium where therespective concentrations C_(A) and C_(B) in the said compounds (A) and(B) belong to the range for formation of a metastable dispersion for theauto-associative system (A+B) used.
 17. The method of claim 16, whereinthe medium obtained at the end of step (E): the sum of theconcentrations C_(A)+C_(B) is between 0.1 and 20 g/l; and the molarratio of the total quantity of cyclodextrin units present within thepolymers (A) introduced, relative to the total quantity of aliphaticchains present as substituents on the polysaccharide macromolecules (B)introduced is between 1:3 and 3:1, and preferably between 1.2 and 2.1.18. The method of claim 16, wherein the concentration of the solution(S_(A)) is between 0.01 g/l and 20 g/l, the concentration of thesolution (S_(B)) is between 0.01 g/l and 20 g/l, and the ratio of thetotal volume of solution (S_(A)) introduced to the total volume ofsolution (S_(B)) introduced is between 1:9 and 9:1.
 19. A method ofpreparation of a composition of claim 12, which consists of placing thesaid compound (C) in contact with a composition comprising an aqueousdispersion of particles (p) of mean hydrodynamic diameter between 50 and5000 nm, wherein said particles contain, in association: (A) polymersbased on cyclodextrin units, with an average content of at least 4cyclodextrin units within their structure; and (B) macromolecules ofpolysaccharides comprising groups G capable of forming inclusioncomplexes with the cyclodextrins present in the structure of the saidpolymers (A), with an average number of groups G per polysaccharidemacromolecule at least equal to 3, wherein said compounds (A) and (B)are water-soluble in the isolated state.
 20. A method of preparation ofa composition as claimed in claim 1 wherein the particles (p) compriseat least one additional chemical compound (C) other than the compounds(A) and (B) comprising a step which consists of effecting a mixture ofan aqueous solution (S_(A)) comprising polymers (A) and the saidadditional compound (C) and an aqueous solution (S_(B)) comprisingmodified polysaccharides (B), the volumes and the concentrations of thesaid solutions (S_(A)) and (S_(B)) being chosen in such a way as toobtain, after the mixing, an aqueous medium where the respectiveconcentrations C_(A) and C_(B) in the said compounds (A) and (B) belongto the range for formation of a metastable dispersion for theauto-associative system (A+B) used.
 21. A method making use of thecomposition according to claim 1 for achieving encapsulation of chemicalcompounds.
 22. A method making use of the composition according to claim12 for achieving a progressive release of compounds (C) present withinthe particles (p) within a medium into which the said composition isintroduced, or in order to limit the contact between the said compounds(C) and the said medium.
 23. A method making use of the composition ofclaim 15, where the compound (C) is an active compound by way of amedicament, for the manufacture of a pharmaceutical composition intendedto deliver the said compound (C) in a progressive manner and/or todeliver this compound (C) in a selective manner at the level of a givenmucous membrane.
 24. A composition obtainable by a lyophilisation of acomposition according to claim 1.